Bladder reconstruction

ABSTRACT

The invention is directed to methods and devices for the reconstruction, repair, augmentation or replacement of laminarily organized organs or tissue structures in a patient in need of such treatment. The device comprises a biocompatible synthetic or natural polymeric matrix shaped to conform to at least a part of the luminal organ or tissue structure with a first cell population on or in a first area and a second cell population such as a smooth muscle cell population in a second area of the polymeric matrix. The method involves grafting the device to an area in a patient in need of treatment. The polymeric matrix comprise a biocompatible and biodegradable material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/600,455, now U.S. Pat. No. 6,576,019, which is the National Stage ofInternational Application No. PCT/US98/22962, filed Oct. 30, 1998 whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication 60/063,790, filed Oct. 31, 1997.

BACKGROUND

1. Field of the Invention

The invention is directed to methods and materials for tissuereconstruction, repair augmentation and replacement, and particularly touse of such treatments in patients having a defect in urogenital tissuessuch as the bladder.

2. Description of the Background

The medical community has directed considerable attention and effort tothe substitution of defective organs with operationally effectivereplacements. The replacements have ranged from completely syntheticdevices such as artificial hearts to completely natural organs fromanother mammalian donor. The field of heart transplants has beenespecially successfully with the use of both synthetic hearts to naturalhearts from living donors. Equal success has not been achieved in manyother organ fields particularly in the field of bladder reconstruction.

The human urinary bladder is a musculomembranous sac, situated in theanterior part of the pelvic cavity, that serves as a reservoir forurine, which it receives through the ureters and discharges through theurethra. In a human the bladder is found in the pelvis behind the pelvicbone (pubic symphysis) and a drainage tube, called the urethra, thatexits to the outside of the body. The bladder, ureters, and urethra areall similarly structured in that they comprise muscular structures linedwith a membrane comprising urothelial cells coated with mucus that isimpermeable to the normal soluble substances of the urine. The trigoneof the bladder, also called the trigonum vesicae, is a smooth triangularportion of the mucous membrane at the base of the bladder. The bladdertissue is elastic and compliant. That is, the bladder changes shape andsize according to the amount of urine it contains. A bladder resembles adeflated balloon when empty but becomes somewhat pear-shaped and risesinto the abdominal cavity when the amount of urine increases.

The bladder wall has three main layers of tissues: the mucosa,submucosa, and detrusor. The mucosa, comprising urothelial cells, is theinnermost layer and is composed of transitional cell epithelium. Thesubmucosa lies immediately beneath the mucosa and its basement membrane.It is composed of blood vessels which supply the mucosa with nutrientsand the lymph nodes which aid in the removal of waste products. Thedetrusor is a layer of smooth muscle cells which expands to store urineand contracts to expel urine.

The bladder is subjected to numerous maladies and injuries which causedeterioration in patients. For example, bladder deterioration may resultfrom infectious diseases, neoplasms and developmental abnormalities.Further, bladder deterioration may also occur as a result of trauma suchas, for example, car accidents and sports injury.

Although a large number of bio-materials, including synthetic andnaturally-derived polymers, have been employed for tissue reconstructionor augmentation (see, e.g., “Textbook or Tissue Engineering” Eds. Lanza,R., Langer, R, and Chick, W, ACM Press, Colorado (1996) and referencescited therein), no material has proven satisfactory for use in bladderreconstruction. For example, synthetic biomaterials such as polyvinyland gelatin sponges, polytetrafluoroethylene (Teflon) felt, and silasticpatches have been relatively unsuccessful, generally due to foreign bodyreactions (see, e.g., Kudish, H. G., J. Urol. 78:232 (1957); Ashkar, L.and Heller, E., J. Urol. 98:91 (1967); Kelami, A. et al., J. Urol.104:693 (1970)). Other attempts have usually failed due to eithermechanical, structural, functional, or biocompatibility problems.Permanent synthetic materials have been associated with mechanicalfailure and calculus formation.

Naturally-derived materials such as lyophilized dura, de-epithelializedbowel segments, and small intestinal submucosa (SIS) have also beenproposed for bladder replacement (for a general review, see Mooney, D.et al., “Tissue Engineering: Urogenital System” in “Textbook of TissueEngineering” Eds. Lanza, R., Langer, R, and Chick, W., ACM Press,Colorado (1996)). However, it has been reported that bladders augmentedwith dura, peritoneum, placenta and fascia contract over time (Kelami,A. et al., J. Urol. 105:518 (1971)). De-epithelized bowel segmentsdemonstrated an adequate urothelial covering for use in bladderreconstruction, but difficulties remain with either mucosal regrowth,segment fibrosis, or both. It has been shown that de-epithelization ofthe intestinal segments may lead to mucosal regrowth whereas removal ofthe mucosa and submucosa may lead to retraction of the intestinalsegment (see, e.g., Atala, A., J. Urol. 156:338 (1996)).

Other problems have been reported with the use of certaingastrointestinal segments for bladder surgery including stone formation,increased mucus production, neoplasia, infection, metabolicdisturbances, long term contracture and resorption. These attempts withnatural or synthetic materials have shown that bladder tissue, with itsspecific muscular elastic properties and urothelial permeabilityfunctions, cannot be easily replaced.

Due to the multiple complications associated with the use ofgastrointestinal segments for bladder reconstruction, investigators havesought alternate solutions. Recent surgical approaches have relied onnative urological tissue for reconstruction, including auto-augmentationand ureterocystoplasty. However, autoaugmentation has been associatedwith disappointing long-term results and ureterocystoplasty is limitedto cases in which a dilated ureter is already present. A system ofprogressive dilation for ureters and bladders has been proposed,however, this has not yet been attempted clinically. Sero-musculargrafts and de-epithelialized bowel segments, either alone or over anative urothelium, have also been attempted. However, graft shrinkageand re-epithelialization of initially de-epithelialized bowel segmentshas been a recurring problem.

One significant limitation besetting bladder reconstruction is directlyrelated to the availability of donor tissue. The limited availability ofbladder tissue prohibits the frequent routine reconstruction of bladderusing normal bladder tissue. The bladder tissue that is available, andconsidered usable, may itself include inherent imperfections anddisease. For example, in a patient suffering from bladder cancer, theremaining bladder tissue may be contaminated with metastasis.Accordingly, the patient is predestined to less than perfect bladderfunction.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies for reconstruction repair ofaugmentation and replacement of luminal organs and tissue structures.

One embodiment of this invention is directed to a method for thereconstruction, repair, augmentation or replacement of laminarilyorganized luminal organs or tissue structures in a patient in need ofsuch treatment. The method involves providing a biocompatible syntheticor natural polymeric matrix shaped to conform to at least a part of theluminal organ or tissue structure in need of said treatment, depositinga first cell population on or in a first area of said polymeric matrix,depositing a second cell population of a different cell type than saidfirst cell population in a second area of the polymeric matrix. Thesecond area is substantially separated from the first area. The shapedpolymeric matrix cell construct is implanted into the patient at thesite in need of treatment to form a laminarily organized luminal organor tissue structure. Another embodiment of this invention is directed toa device for the reconstruction, repair, augmentation or replacement ofluminarily organized luminal organs or tissue structures. The devicecomprises an implantable, biocompatible, synthetic or natural polymericmatrix with at least two separate surfaces. The polymeric matrix isshaped to conform to a at least a part of the luminal organ or tissuestructure in need of said treatment and at least two different cellpopulations are deposited in substantially separate areas either on orin the polymeric matrix to form a luminarily organized matrix/cellconstruct.

A further embodiment of this invention is directed to a device for therepair, reconstruction, augmentation or replacement of damaged ormissing bladder tissue in a patient in need of such treatment. Thedevice comprises an implantable, biocompatible synthetic or naturalpolymeric matrix which is shaped to conform to the part of a bladdertissue in need of treatment. Urothelial cells are deposited on and nearthe inside surface of the matrix, and smooth muscle cells are depositedon and near the outside surface of said matrix. Upon implantation intothe patient, the device forms a laminarily organized luminal tissuestructure with the compliance of normal bladder tissue.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description and may be learned from the practice of theinvention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts (A) a native canine bladder prior to trigone-sparingcystectomy; (B) an engineered neo-Organ anastomosed to the trigone; and(C) an implant, decompressed by a transurethral and suprapubic catheter,wrapped with omentum.

FIG. 2 depicts (A) bladder capacities and (B) compliance at differentpostoperative time points relative to preoperative capacities of 100%.

FIG. 3 depicts radiographic cystograms 11 months after subtotalcystectomy followed by (A) Subtotal cystectomy without reconstruction(Group A); (B) Polymer only implant (Group B); and (C) tissue engineeredneo-organ (Group C).

FIG. 4 depicts (A and B) gross aspect of subtotal cystectomy control; (Cand D) polymer only implant; and (E and F) tissue engineered neo-organretrieved after 11 months.

FIG. 5 depicts H&E histological results six months after surgery of (A)normal canine bladder; (B) bladder dome of the cell-free polymerreconstructed bladder (group B); (C) the tissue engineered neoorgan(group C).

FIG. 6 depicts positive immunocytochemical staining of tissue engineeredneo-organ for (A) pancytokeratins AE1/AE3; (B) Urothelialdifferentiation related membrane proteins; (C) smooth muscle actin; and(D) S-100 antibodies six months after implantation.

DESCRIPTION OF THE INVENTION

The present invention provides methods and devices that facilitatetissue reconstruction. In its broadest form, the methods and devices ofthe present invention are useful in the reconstruction, repair,augmentation or replacement of organs or tissues structures thatcomprise multilayer cellular organization and particularly those organsor tissue structures that are luminal in nature. More particularly, thepresent invention provides methods and devices that facilitate thereconstruction, repair, augmentation or replacement of shaped holloworgans or tissue structures that exhibit a laminar segregation ofdifferent cell types and that have a need to retain a general luminalshape. Luminal organs or tissue structures that contain a smooth musclecell (SMC) layer to impart compliant or contractible properties to theorgan or structure are particularly well suited to the methods anddevices of the present invention.

In an example of one preferred embodiment of the invention, the luminalorgan is the bladder, which has an inner layer of a first cellpopulation that comprises urothelial cells and an outer layer of asecond cell population that comprises smooth muscle cells. Thisorganization is also present in other genitourinary organs and tissuestructures such as the ureters and urethra. Laminarily organized organsor tissues refer to any organ or tissue made up of; or arranged inlaminae including ductal tissue. Other suitable laminarily organizedluminal organs, tissue structure, or ductal tissues to which the presentinvention is directed include vas deferens, fallopian tubes, lacrimalducts, trachea, stomach, intestines, vasculature, biliary duct, ductusejaculatorius, ductus epididymidis, ductus parotideus, and surgicallycreated shunts.

The method of the present invention in its broadest aspect encompassesas a first step providing a biocompatible synthetic or natural polymericmatrix that is shaped to conform to its use as a part or all of theluminal organ or tissue structure to be repaired, reconstructed,augmented or replaced. A biocompatible material is any substance nothaving toxic or injurious effects on biological function. The shapedmatrix is preferably porous to allow for cell deposition both on and inthe pores of the matrix. The shaped polymeric matrix is then contacted,preferably sequentially, With at least two different cell populationssupplied to separate areas of the matrix (e.g., inside and outside) toseed the cell population on and/or into the matrix. The seeded matrix isthen implanted in the body of the recipient where the separate,laminarily organized cell populations facilitate the formation ofneo-organs or tissue structures.

In a preferred embodiment, the materials and methods of the inventionare useful for the reconstruction or augmentation of bladder tissue.Thus, the invention provides treatments for such conditions as bladderexstrophy, bladder volume insufficiency, reconstruction of bladderfollowing partial or total cystectomy, repair of bladders damaged bytrauma, and the like.

While reference is made herein to augmentation of bladder according tothe invention, it will be understood that the methods and materials ofthe invention are useful for tissue reconstruction or augmentation of avariety of tissues and organs in a subject. Thus, for example, organs ortissues such as bladder, ureter, urethra, renal pelvis, and the like,can be augmented or repaired with polymeric matrixes seeded with cells.The materials and methods of the invention further can be applied to thereconstruction or augmentation of vascular tissue (see, e.g., Zdrahala,R. J., J Biomater. Appl. 10 (4): 309-29 (1996)), intestinal tissues,stomach (see, e.g., Laurencin, C.T. et al., J Biomed Mater. Res. 30 (2):133-8 1996), and the like. The patient to be treated may be of anyspecies of mammals such as a dog, cat, pig, horse, cow, or human, inneed of reconstruction, repair, or augmentation of a tissue.

Polymeric matrices Biocompatible material and especially biodegradablematerial is the preferred material for the construction of the polymericmatrix. The polymeric matrix is used in the construction of thereconstructive urothelial graft (RUG). The RUG is an implantable,biocompatible, synthetic or natural polymeric matrix with at least twoseparate surfaces. The RUG is shaped to conform to a at least a part ofthe luminal organ or tissue structure in need or treatment and has atleast two different cell populations deposited in substantially separateareas either on or in the polymeric matrix. Thus the RUG is a laminarilyorganized matrix/cell construct.

Biocompatible refers to materials which do not have toxic or injuriouseffects on biological functions. Biodegradable refers to material thatcan be absorbed or degraded in a patient's body. Examples ofbiodegradable materials include, for example, absorbable sutures.Representative materials for forming the biodegradable structure includenatural or synthetic polymers, such as, for example, collagen,poly(alpha esters) such as poly(lactate acid), poly(glycolic acid),polyorthoesters and polyanhydrides and their copolymers, which degradedby hydrolysis at a controlled rate and are reabsorbed. These materialsprovide the maximum control of degradability, manageability, size andconfiguration. Preferred biodegradable polymer material includepolyglycolic acid and polyglactin, developed as absorbable syntheticsuture material. Polyglycolic acid and polyglactin fibers may be used assupplied by the manufacturer. Other biodegradable materials includecellulose ether, cellulose, cellulosic ester, fluorinated polyethylene,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, or copolymers or physical blends of thesematerials. The material may be impregnated with suitable antimicrobialagents and may be colored by a color additive to improve visibility andto aid in surgical procedures.

A presently preferred biocompatible polymer is Polyglactin, developed asabsorbable synthetic suture material, a 90:10 copolymer of glycolide andlactide, manufactured as Vicryl<< braided absorbable suture (EthiconCo., Somerville, N.J.) (Craig P. H., Williams J. A., Davis K. W., etal.: A Biological Comparison of Polyglactin 910 and Polyglycolic AcidSynthetic Absorbable Sutures. Surg. 141; 1010, (1975)) and polyglycolicacid. Polyglactin and polyglycolic acid fibers can be used as suppliedby the manufacturer. The biocompatible polymer may be shaped usingmethods such as, for example, solvent casting, compression molding,filament drawing, meshing, leaching, weaving and coating. In solventcasting, a solution of one or more polymers in an appropriate solvent,such as methylene chloride, is cast as a branching pattern reliefstructure. After solvent evaporation, a thin film is obtained. Incompression molding, a polymer is pressed at pressures up to 30,000pounds per square inch into an appropriate pattern. Filament drawinginvolves drawing from the molten polymer and meshing involves forming amesh by compressing fibers into a felt-like material. In leaching, asolution containing two materials is spread into a shape close to thefinal form of the RUG. Next a solvent is used to dissolve away one ofthe components, resulting in pore formation. (See Mikos, U.S. Pat. No.5,514,378, hereby incorporated by reference). In nucleation, thin filmsin the shape of a RUG are exposed to radioactive fission products thatcreate tracks of radiation damaged material. polycarbonate sheets areetched with acid or base, turning the tracks of radiation damagedmaterial into pores. Finally, a laser may be used to shape and burnindividual holes through many materials to form a RUG structure withuniform pore sizes. Coating refers to coating or permeating a polymericstructure with a material such as, for example liquefied copolymers(poly-DL-lactide co-glycolide 50:50 80 mg/ml methylene chloride) toalter its mechanical properties. Coating may be performed in one layer,or multiple layers until the desired mechanical properties are achieved.These shaping techniques may be employed in combination, for example, apolymeric matrix may be weaved, compression molded and glued together.Furthermore different polymeric materials shaped by different processesmay be joined together to form a composite shape. The composite shapemay be a laminar structure. For example, a polymeric matrix may beattached to one or more polymeric matrixes to form a multilayerpolymeric matrix structure. The attachment may be performed by gluingwith a liquid polymer or by suturing. In addition, the polymeric matrixmay be formed as a solid block and shaped by laser or other Standardmachining techniques to its desired final form. Laser shaping refers tothe process of removing materials using a laser.

The biodegradable polymers can be characterized with respect tomechanical properties, such as tensile strength using an Instron tester,for polymer molecular weight by gel permeation chromatography (GPC),glass, transition temperature by differential scanning calorimetry (DSC)and bond structure by infrared (IR) spectroscopy; with respect totoxicology by initial screening tests involving Ames assays and in vitroteratogenicity assays and implantation studies in animals forimmunogenicity, inflammation, release and degradation studies. In vitrocell attachment and viability can be assessed using scanning electronmicroscopy, histology and quantitative assessment with radioisotopes.The biodegradable material may also be characterized with respect to theamount of time necessary for the material to degrade when implanted in apatient. By varying the construction, such as, for example, thethickness and mesh size, the biodegradable material may substantiallybiodegrade between about 2 years or about 2 months, preferably betweenabout 18 months and about 4 months, most preferably between about 15months and about 8 months and most preferably between about 12 monthsand about 10 months. If necessary, the biodegradable material may beconstructed so as not to degrade substantially within about 3 years, orabout 4 years or about five or more years.

The polymeric matrix may be fabricated with controlled pore structure asdescribed above. The size of the pores may be used to determine the celldistribution. For example, the pores on the polymeric matrix may belarge to enable cells to migrate from one surface to the oppositesurface. Alternatively, the pores may be small such that there is fluidcommunication between the two sides of the polymeric matrix but cellscannot pass through. Suitable pore size to accomplish this objective maybe about 0.04 micron to about 10 microns in diameter, preferably betweenabout 0.4 micron to about 4 microns in diameter. In some embodiments,the surface of the polymeric matrix may comprise pores sufficientlylarge to allow attachment and migration of a first population of cellsinto the pores. The pore size may be reduced in the interior of thepolymeric matrix to prevent cells from migrating from one side of thepolymeric matrix to the opposite side. On the opposite side of thepolymeric matrix, the pores may again enlarge to allow the attachmentand establishment of a second population of cells. Because of thereduced pore size in the interior of the polymeric matrix, the firstcell population and the second cell population initially cannot mix. Oneembodiment of a polymeric matrix with reduced pore size is a laminatedstructure of a small pore material sandwiched between two large porematerial. Alternatively, a large pore material laminated to a small porematerial may also allow cells to establish growth on both sides withoutany intermixing of cells. Polycarbonate membranes are especiallysuitable because they can be fabricated in very controlled pore sizessuch as, for example, about 0.01 microns, about 0.05 micron, about 0.1micron, about 0.2 micron, about 0.45 micron, about 0.6 micron, about 1.0micron, about 2.0 microns and about 4.0 microns. At the submicron levelthe polymeric matrix may be impermeable to bacteria, viruses and othermicrobes. At the present time, a mesh-like structure formed of fibers,which may be round, scalloped, flattened, star shaped, solitary orentwined with other fibers is preferred. The use of branching fibers isbased upon the same principles which nature has used to solve theproblem of increasing surface area proportionate to volume increases.All multicellular organisms utilize this repeating branching structure.Branching systems represent communication networks between organs, aswell as the functional units of individual organs. Seeding andimplanting this configuration with cells allows implantation of largenumbers of cells, each of which is exposed to the environment of thehost, providing for free exchange of nutrients and waste whileneovascularization is achieved. The polymeric matrix may be madeflexible or rigid, depending on the desired final form, structure andfunction.

In one preferred embodiment, the polymeric matrix is formed with apolyglycolic acid with an average fiber diameter of 15 μm and configuredinto a bladder shaped mold using 4-0 polyglactin 910 sutures. Theresulting structure is coated with a liquefied copolymer, such as, forexample, pol-DL-lactide-co-glycolide 50:50, 80 milligram per millilitermethylene chloride, in order to achieve adequate mechanicalcharacteristics and to set its shape.

Polymeric matrixes can be treated with additives or drugs prior toimplantation (before or after the polymeric matrix is seeded with cells,if the optional seeded cells are employed), e.g., to promote theformation of new tissue after implantation. Thus, for example, growthfactors, cytokines, extracellular matrix components, and other bioactivematerials can be added to the polymeric matrix to promote graft healingand formation of new tissue. Such additives will in general be selectedaccording to the tissue or organ being reconstructed or augmented, toensure that appropriate new tissue is formed in the engrafted organ ortissue (for examples of such additives for use in promoting bonehealing, see, e.g., Kirker-Head, C.A. Vet. Surg. 24 (5): 408-19 (1995)).For example, when polymeric matrixes (optionally seeded with endothelialcells) are used to augment vascular tissue, vascular endothelial growthfactor (VEGF), (see, e.g., U.S. Pat. No. 5,654,273) can be employed topromote the formation of new vascular tissue. Growth factors and otheradditives (e.g., epidermal growth factor (EGF), heparin-bindingepidermal-like growth factor (HBGF), fibroblast growth factor (FGF),cytokines, genes, proteins, and the like) can be added in amounts inexcess of any amount of such growth factors (if any) which may beproduced by the cells seeded on the polymeric matrix, if added cells areemployed. Such additives are preferably provided in an amount sufficientto promote the formation of new tissue of a type appropriate to thetissue or organ, which is to be repaired or augmented ( e.g., by causingor accelerating infiltration of host cells into the graft). Other usefuladditives include antibacterial agents such as antibiotics.

One preferred supporting matrix is composed of crossing filaments whichcan allow cell survival by diffusion of nutrients across short distancesonce the cell support matrix is implanted. The cell support matrixbecomes vascularized in concert with expansion of the cell massfollowing implantation.

The building of three-dimensional structure constructs in vitro, priorto implantation, facilitates the eventual terminal differentiation ofthe cells after implantation in vivo, and minimizes the risk of aninflammatory response towards the matrix, thus avoiding graftcontracture and shrinkage.

The polymeric matrix may be shaped into any number of desirableconfigurations to satisfy any number of overall system, geometry orspace restrictions. For example, in the use of the polymeric matrix forbladder reconstruction, the matrix may be shaped to conform to thedimensions and shapes of the whole or a part of a bladder. Naturally,the polymeric matrix may be shaped in different sizes and shapes toconform to the bladders of differently sized patients. Optionally, thepolymeric matrix should be shaped such that after its biodegradation,the resulting reconstructed bladder may be collapsed when empty in afashion similar to a natural bladder. The polymeric matrix may also beshaped in other fashions to accommodate the special needs of the .patient. For example, a previously injured or disabled patient, may havea different abdominal cavity and may require a bladder reconstructed toadapt to fit. In other embodiments of the invention, the polymericmatrix is used for the treatment of laminar structures in the body suchas urethra, vas deferens, fallopian tubes, lacrimal ducts. In thoseapplications the polymeric matrix may be shaped as a hollow tube.

The polymeric matrix may be sterilized using any known method beforeuse. The method used depend on the material used in the polymericmatrix. Examples of sterilization methods include steam, dry heat,radiation, gases such as ethylene oxide, gas and boiling.

Harvesting Cells for the Reconstructive Urothelial Graft (RUG)

The RUG is constructed in part using urothelial cells and smooth musclecells from a donor. One advantage of the methods of the invention isthat because of the rapid growth of the urothelial and smooth musclecells, sufficient cells for the construction of a RUG may be grown inless than 5 weeks. In an autologous RUG, the cells may be derived fromthe patient's own tissue such as, for example, from the bladder,urethra, ureter, and other urogenital tissue. In an allogeneic RUG, thecells may be derived from other member of the patient's species. In axenogenic RUG, the cells may be derived from a species different fromthe patient. Donor cells may be from any urothelial cells and smoothmuscle cells origin and from any mammalian source such as, for example,humans, bovine; porcine, equine, caprine and ovine sources. Urothelialcells and smooth muscle cells may be isolated in biopsies, or autopsies.In addition, the cells may be frozen or expanded before use.

To prepare for RUG construction, tissue containing urothelial and smoothmuscle cells is dissociated separately into two cell suspensions.Methods for the isolation and culture of cells were discussed in issuedU.S. Pat. No. 5,567,612 which is herein specifically incorporated byreference. Dissociation of the cells to the single cell stage is notessential for the initial primary culture because single cell suspensionmay be reached after a period, such as, a week, of in vitro culture.Tissue dissociation may be performed by mechanical and enzymaticdisruption of the extracellular matrix and the intercellular junctionsthat hold the cells together. Urothelial cells and smooth muscle cellsfrom all developmental stages, such as, fetal, neonatal, juvenile toadult may be used.

Cells (such as autologous cells) can be cultured in vitro, if desired,to increase the number of cells available for seeding on the polymericmatrix “scaffold.” The use of allogenic cells, and more preferablyautologous cells, is preferred to prevent tissue rejection. However, ifan immunological response does occur in the subject after implantationof the RUG, the subject may be treated with immunosuppressive agentssuch as, for example, cyclosporin or FK506, to reduce the likelihood ofrejection of the RUG. In certain embodiments, chimeric cells, or cellsfrom a transgenic animal, can be seeded onto the polymeric matrix.

Cells may be transfected prior to seeding with genetic material. Usefulgenetic material may be, for example, genetic sequences which arecapable of reducing or eliminating an immune response in the host. Forexample, the expression of cell surface antigens such as class I andclass II histocompatibility antigens may be suppressed. This may allowthe transplanted cells to have reduced chance of rejection by the host.In addition, transfection could also be used for gene delivery.Urothelial and muscle cells could be transfected with specific genesprior to polymer seeding. The cell-polymer construct could carry geneticinformation required for the long term survival of the host or thetissue engineered neo-organ. For example, cells may be transfected toexpress insulin for the treatment of diabetes.

Cell cultures may be prepared with or without a cell fractionation step.Cell fractionation may be performed using techniques, such as florescentactivated cell sorting, which is known to those of skill in the art.Cell fractionation may be performed based on cell size, DNA content,cell surface antigens, and viability. For example, urothelial cells maybe enriched and smooth muscle cells and fibroblast cells may be reducedfor urothelial cell collection. Similarly, smooth muscle cells may beenriched and urothelial cells and fibroblast cells may be reduced forsmooth muscle cell collection. While cell fractionation may be used, itis not necessary for the practice of the invention.

Cell fractionation may be desirable, for example, when the donor hasdiseases such as bladder cancer or metastasis of other tumors to thebladder. A bladder cell population may be sorted to separate malignantbladder cells or other tumor cells from normal noncancerous bladdercells. The normal noncancerous bladder cells, isolated from one or moresorting techniques, may then be used for bladder reconstruction.

Another optional procedure in the method is cryopreservation. Cryogenicpreservation may be useful, for example, to reduce the need for multipleinvasive surgical procedures. Cells taken from a bladder may beamplified and a portion of the amplified cells may be used and anotherportion may be cryogenically preserved. The ability to amplify andpreserve cells allow considerable flexibility in the choice of donorcells. For example, cells from a histocompatible donor, may be amplifiedand used in more than one recipient.

Another example of the utility of cryogenic preservation is in tissuebanks. Donor cells may be cryopreserved along with histocompatibilitydata. Donor cells may be stored, for example, in a donor tissue bank. Astissue is needed for bladder reconstruction, cells may be selected whichare most histocompatible to the patient. Patients who have a disease orundergoing treatment which may endanger their bladders may cryogenicallypreserve a biopsy of their bladders. Later, if the patient's own bladderfails, the cryogenically preserved bladder cells may be thawed and usedfor treatment. For example, if bladder cancer reappeared after bladderreconstruction, cryogenically preserved cells may be used for bladderreconstruction without the need isolate more tissue from the patient forculture.

Seeding

Seeding of cells onto the polymeric matrix can be performed, e.g., as isdescribed in the Example or according to standard methods. For example,the seeding of cells onto polymeric substrates for use in tissue repairhas been reported (see, e.g., Atala, A. et al., J. Urol. 148 (2 Pt 2):658-62 (1992); Atala, A., et al. J. Urol. 150 (2 Pt 2): 608-12 (1993)).Cells grown in culture can be trypsinized to separate the cells, and theseparated cells can be seeded on the polymeric matrix. Alternatively,cells obtained from cell culture can be lifted from a culture plate as acell layer, and the cell layer can be directly seeded onto the polymericmatrix without prior separation of the cells.

In a preferred embodiment, at least 50 million cells are suspended inmedia and applied to each square centimeter of a surface of a polymericmatrix. The polymeric matrix is incubated under standard culturingconditions, such as, for example, 37° 5% CO₂, for a period of time untilthe cells attached. However, it will be appreciated that the density ofcells seeded onto the polymeric substrate can be varied. For example,greater cell densities promote greater tissue formation by the seededcells, while lesser densities may permit relatively greater formation oftissue by cells infiltrating the graft from the host. Other seedingtechniques may also be used depending on the polymeric matrix and thecells. For example, the cells may be applied to the polymeric matrix byvacuum filtration. Selection of cell types, and seeding of cells onto apolymeric matrix, will be routine to one of ordinary skill in the art inlight of the teachings herein.

In an embodiment of the invention, a polymeric matrix is seeded on twosides with two different populations of cells. This may be performed byfirst seeding one side of the polymeric matrix and then seeding theother side. For example, the polymeric matrix may be placed with oneside on top and seeded. Then the polymeric matrix may be repositioned sothat a second side is on top. The second side may then be seeded with asecond population of cells. Alternatively, both sides of the polymericmatrix may be seeded at the same time. For example, two cell chambersmay be positioned on both sides (i.e., a sandwich) of the polymericmatrix. The two chambers may be filled with different cell populationsto seed both sides of the polymeric matrix simultaneously. Thesandwiched polymeric matrix may be rotated, or flipped frequently toallow equal attachment opportunity for both cell populations:simultaneous seeding may be preferred when the pores of the polymericmatrix are sufficiently large for cell passage from one side to theother side. Seeding the polymeric matrix on both sides simultaneouslywill reduce the likelihood that the cells would migrate to the oppositeside.

In another embodiment of the invention, two separate polymeric matrixesmay be seeded with different cell populations. After seeding, the twomatrixes may be attached together to form a single polymeric matrix withtwo different cell populations on the two sides. Attachment of thematrixes to each other may be performed using standard procedures suchas fibrin glue, liquid co-polymers, sutures and the like.

Surgical Reconstruction

Grafting of polymeric matrixes to an organ or tissue to be augmented canbe performed according to the methods described in the Examples oraccording to art-recognized methods. As shown in the examples, thepolymeric matrix can be grafted to an organ or tissue of the subject bysuturing the graft material to the target organ.

The techniques of the invention may also be used to treat cancer of thebladder. For example, a normal bladder tissue sample may be excised froma patient suffering from bladder cancer. Urothelial cells and smoothmuscle cells from the tissue sample may be cultured for a period of timein vitro and expanded. The cells may be sorted using a florescentactivated cell sorter to remove cancerous or precancerous cells. Thesorted cells may be used to construct a RUG. At the same time, thepatient may be treated for cancer. Cancer treatment may involve excisionof the cancerous part of the bladder in addition to chemotherapy orradiation treatment. After the cancer treatment, the RUG may be used toreconstruct the bladder.

While a method for bladder reconstruction is disclosed in the Example,other methods for attaching a graft to an organ or tissue of the subject(e.g., by use of surgical staples) may also be employed. Such surgicalprocedures can be performed by one of ordinary skill in the artaccording to known procedures.

As a result of these benefits, the present method of bladderreconstructive surgery is suitable for bladder tissue repair undernumerous circumstances. As described above the bladder graft may be usedto repair a deteriorated bladder due to.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description and may be learned from practice of the invention.

EXAMPLES Example 1

Creation of Bladder-Shaped Polymers

A synthetic polymer matrix of polyglycolic acid with an average fiberdiameter of about 15 μm and an interfiber distance between about 0 toabout 200 μm and dimensions of about 10 cm by about 10 cm was configuredinto a bladder shaped mold using biodegradable 4-0 polyglactin 910sutures. The resulting flexible scaffold was coated with a liquefiedcopolymer, a mixture of about 50% poly-DL-lactide-coglycolide and about50% 80 mg/ml methylene chloride, in order to achieve adequate mechanicalcharacteristics. After sterilization with ethylene oxide, the polymerswere stored in a desiccator.

Example 2

Cell Harvest and Culture

A total of 14 beagle dogs underwent a trigone-sparing cystectomy. Theanimals were randomly assigned to one of three groups. Two were assignedto Group A and underwent closure of the trigone without a reconstructiveprocedure. Six were assigned to Group B and underwent bladderreconstruction with a cell-free bladder shaped biodegradable polymer.Six were assigned to Group C and underwent bladder reconstruction usinga prefabricated tissue engineered neo-organ. The neo-organ comprises abladder shaped biodegradable polymer with autologous urothelial cellsattached to the luminal surface and smooth muscle cells attached to theexterior surface. The cell populations had been separately expanded froma previously harvested autologous transmural bladder specimen.

The six animals in group C, which were to be reconstructed with a tissueengineered neo-organ, underwent a transmural bladder biopsy of about onesquare centimeter which was harvested from the vesical dome via aminimal suprapubic midline incision under general anesthesia. The defectwas closed with a 4-0 polyglactin 910 suture. The bladder specimens werekept in prewarmed keratinocyte medium, and cell harvest for in-vitrocultures was initiated immediately after tissue excision.

Urothelial and smooth muscle cell populations, dissociated from the onesquare centimeter bladder biopsies, could be routinely expanded andpassaged separately. The average time elapsed between the initialbladder biopsy and final implantation of the tissue engineeredneo-organs was 32+/−2.8 days (Mean+SD). Approximately thirty-two 25 cmplates of each cell type, muscle and urothelial cells, containingapproximately 10⁷ cells per plate, were processed to constitute onetissue engineered neo-organ.

The harvested cells were cultured according to previously publishedprotocols of Atala et al., (J. Urol. 150: 608, 1993) and Cilento et al.,(J. Urol. 152: 655, 1994.) which are herein specifically incorporated byreference. The urothelial and muscular layers of the bladder biopsy weremicrosurgically detached from each other and processed separately.Briefly, the dissected smooth muscle tissue was cut into cubes of aboutone millimeter and primarily plated on a 10 cm tissue culture petridish. Smooth muscle cultures were maintained and expanded withDulbeccos's Modified Eagles Medium (DMEM, Sigma, St. Louis, Mo.)supplemented with 10% fetal calf serum (Biowhittaker Inc., Walkersville,Md.). Urothelial cells were also dissected into one millimeter cubes andplated on 24 well plates. Urothelial cultures were maintained andexpanded with serum-free keratinocyte growth medium supplemented withabout 5 ng/ml of epidermal growth factor and about 50 μg/ml of bovinepituitary extract (Gibco BRL, Life Technologies, Grand Island, N.Y.).All cell cultures were incubated at 37° C. in a humidified atmospheremaintained at about 5% level of carbon dioxide. Medium was changed twiceweekly. For cell passage cultures at about 80% confluence weretrypsinized by incubation for 5 minutes in 0.05% trypsin in 1 millimoleethylenediaminetetraacetic acid. After this period soybean trypsininhibitor, at 2 units per unit of trypsin, was added to the cellsuspension. Both urothelial and smooth muscle cells were expandedseparately until sufficient cell quantities were available for a seedingdensity of approximately one million cells per square centimeter ofpolymer surface.

Example 3

Cell Seeding on Polymer Scaffold

For each tissue engineered neo-organ, about 32 confluent 25 cm plates ofeach cell type, muscle and urothelium, were processed for seeding.Muscle cell cultures were trypsinized, collected, washed and combined inone tube. The exterior surface of the pre-molded bladder shaped polymermatrix was seeded with the resuspended smooth muscle cell population.The cell-seeded polymers were incubated in Dulbeccos's Modified EaglesMedium (DMEM, Sigma, St. Louis, Mo.) supplemented with 10% fetal calfserum (Biowhittaker Inc., Walkersville, Md.). The medium was changed at12 hour intervals to ensure sufficient supply of nutrients. After 48hours of incubation, the urothelial cells were processed in a similarfashion and were seeded onto the luminal surface of the polymer.

Example 4

Bladder Reconstruction

Following pretreatment with intramuscular injection of 0.1 mg ofacepromazine for every kilogram of body weight, surgery was performedunder intravenous pentobarbital anesthesia of about 25 to about 35 mgper kilogram of body weight with endotracheal aeration: About 500 mg ofCefazolin sodium was administered intravenously both preoperatively andintraoperatively. Additional treatment of subcutaneously Cefazolinsodium was administered for 5 postoperative days at a dose of about 30milligrams per kilogram body weight per day. Postoperative analgesictreatment was managed with subcutaneous injections of about 0.1 to about0.6 milligrams of butorphanol per kilogram of body weight.

As shown in FIG. 1A, a midline laparotomy was performed, the bladder wasexposed (FIG. 1A) and both ureters were identified. The bladder wall wasincised ventrally and both ureteric junctions were visualized andtemporarily intubated with 4 F stents. A subtotal cystectomy wasperformed, sparing the trigone area bearing the urethra and ureteraljunctions. Care was taken not to compromise or obstruct the ureters. Intwo animals the trigone was closed, without any polymer graft, with twolayers of 4-0 vicryl. As depicted in FIG. 1B, 12 animals undergone ananastomosis between the bladder shaped polymer matrix and the trigonewith interlocking running sutures of 4-0 vicryl. Of the 12 animals, 6received a bladder shaped polymer alone, and six received a bladdershaped polymer coated with cells. A 10 F silicone catheter was insertedinto the urethra from the trigone in a retrograde fashion. An 8 Fsuprapubic catheter was brought into the bladder lumen passing through ashort submucosal tunnel in the trigonal region. The suprapubic catheterwas secured to the bladder serosa with a pursestring suture of 4-0chromic. The anastomosis between trigone and graft was marked at eachquadrant with permanent polypropylene sutures for future graft siteidentification. The neo-bladder was covered with fibrin glue (VitexTechnologies Inc., New York, N.Y.). As depicted in FIG. 1C, omentum waswrapped and secured around the neo-reservoir. The abdomen was closedwith three layers of 3-0 vicryl. After recovery from anesthesia, allanimals wore restraint collars to avoid wound and catheter manipulationduring the early postoperative period. The transurethral catheters wereremoved between postoperative days 4 and 7. Cystograms were performedabout four weeks postoperatively, immediately prior to the suprapubiccatheter removal. Cystograms and urodynamic studies were seriallyperformed at about 1, about 2, about 3, about 4, about 6 and about 11months after surgery.

Example 5

Analysis of Reconstructed Bladder

Urodynamic studies and radiographic cystograms were performedpreoperatively and postoperatively at about 1, about 2, about 3, about4, about 6, and about 11 months after surgery. The two animals whounderwent closure of the trigone without a reconstructive procedure weresacrificed at about 11 months. Animals from the remaining twoexperimental groups were sacrificed at about 1, about 2, about 3, about4, about 6 and about 11 months after surgery. Bladders were retrievedfor gross, histological and immunocytochemical analyses.

Urodynamic studies were performed using a 7 F double-lumen transurethralcatheter. The bladders were emptied and intravesical pressures wererecorded during instillation of prewarmed saline solution at constantrates. Recordings were continued until leak point pressures (LPP) werereached. Bladder volume at capacity (Vol_(max)), LLP and bladdercompliance (Vol_(max)/LLP) were documented. Bladder compliance , alsocalled bladder elastance, denotes the quality of yielding to pressure orforce without disruption. Bladder compliance is also an expression ofthe measure of the ability to yield to pressure or force withoutdisruption, as an expression of the distensibility of the bladder. It isusually measured in units of volume change per unit of pressure change.Subsequently, radiographic cystograms were performed. The bladders wereemptied and contrast medium was instilled intravesically underfluoroscopic control.

Urodynamic Results Prior to trigone-sparing cystectomy, the animals ofgroups A, B and C did not significantly differ in preoperative meanbladder capacity (78+/−16 ml,63 +/−22 ml, 69+/−8 ml, p=0.44, [Means +/−ASD]) or preoperative bladder compliance (2.6+−0.2 ml/cm H₂O, 2.2+/−1.2ml/cm H₂O, 2.1+/−1.1 ml/cm H₂O, p=0.85, [Means +/−SD]).

Both control animals, which did not undergo reconstruction aftersubtotal cystectomy, could only maintain 22% (+/−2%) of the nativecapacity during the observed period. A pattern of frequent voiding wasobvious in these animals. The animals reconstructed with cell-freepolymers developed mean bladder capacities of 46% (+/−20%) ofpreoperative values. A mean bladder capacity of 95% (+/−9%) of theoriginal pre-cystectomy volume was achieved by the tissue engineeredbladder replacements (FIG. 2A).

The subtotal cystectomy bladders which were not reconstructed showed apronounced reduction in bladder compliance to mean values of 10% (+/−3%)of the preoperative values. All polymer only implants without cells alsohad a considerable loss of compliance. At various time points ofsacrifice, bladder compliances were reduced to an average of 42%(+/−21%) of the preoperative values. The compliance of the tissueengineered bladders showed almost no difference from the preoperativevalues measured when the native bladder was present (106% +/−16%, FIG.2B).

Clinically, all animals had a stable course after bladderreconstruction, were able to void spontaneously upon catheter removaland survived their intended study periods. One month after surgery, theradiographic cystograms showed a watertight reservoir in all animals.Cystograms of the subtotal cystectomy only animals showed thatunaugmented trigones were only able to regenerate minimal reservoircapacities throughout the study period. The polymer only implantsdemonstrated signs of partial graft collapse. The tissue engineeredbladders were fully distendable and their contour could be delineatedfrom the native trigone. During follow-up cystograms, the polymer onlyimplants continued to show smaller sized reservoirs while the tissueengineered bladders appeared normal in size and configuration (FIG. 3).

Example 6

Gross Findings

At the intended time points, the animals were euthanized by intravenouspentobarbital administration. The internal organs and the urogenitaltract were inspected for gross abnormalities. The bladder was retrievedand the marking sutures identifying the transition zone between nativetrigone and graft were exposed. Cross sections were taken from withinthe native trigone, the outlined transition zone and the proximallylocated neo-bladder.

Trigone-Sparing Cystectomy only (Group A ). The reservoirs appearedsmall, but normal in appearance (FIGS. 4A and B).

Polymer only Bladders (Group B): Gross inspection of the cell-freepolymer implant retrieved at one month showed that the originalspherical architecture of the polymer had partially collapsed. Asolitary, asymptomatic bladder stone of 11 mm was found in the 2 monthtime point, representing the only incidence of lithogenesis in thisstudy. At the two month time point, graft shrinkage of approximately 50%was macroscopically obvious at necropsy. The bladders retrieved at 4, 6and 11 months contained progressive formations of thick scar tissue atthe dome and were firmly covered with adherent omentum (FIGS. 4C and D).By 11 months, graft shrinkage of over 90% was obvious macroscopically.The initially placed polypropylene marking sutures were noted in thearea of the trigone, adjacent to the scar tissue. Approximately 10% ofthe total bladder area was above the marking sutures.

Tissue Engineered Neo-Organs (Group C): Autopsy exploration showed nosigns of upper tract obstruction, lithogenesis, encrustration or otherabnormalities for all investigated time points. At one month, thepolymer scaffold inside the omentum-wrapped tissue engineeredneo-bladder remained visually and palpably identifiable. Theneo-bladders had a flexible, and distendable configuration. At 6 and 11months, omental adhesions could be bluntly separated from the bladderdome, and a serosa-like layer had regenerated over the tissue engineeredneo-organ (FIG. 4E). The initially placed polypropylene marking sutureswere noted in the distal region of the bladder, at the level of thetrigone. Approximately 70% of the total bladder area was above themarking sutures. Upon entering the bladder ventrally, a smooth mucosalsurface was noted, without any differences between the native and tissueengineered bladder (FIG. 4F).

During the duration of the study, none of the dogs demonstrated anyeffects. All animals survived until the time of sacrifice without anynoticeable complications such as urinary tract infection or calculiformation. Fluoroscopic cystography of all the augmented bladders showeda normal bladder configuration without any leakage at one, two and threemonths after the procedure At retrieval, the augmented bladders appearedgrossly normal without any evidence of diverticular formation in theregion of the graft. The thickness of the grafted segment was similar tothat of the native bladder tissue. There was no evidence of adhesion orfibrosis. Histologically, all retrieved bladders contained a normalcellular organization consisting of a urothelial lined lumen surroundedby submucosal tissue and smooth muscle. An angiogenic response wasevident in all specimens.

Example 7

Histological and Immunocytochemical Findings

Specimens were fixed in 10% buffered formalin and processed. Tissuesections were cut at about 4 to about 6 microns for routine stainingwith Hematoxylin and Eosin (H&E) and Masson's trichrome.Immunocytochemical staining methods were employed with several specificprimary antibodies in order to characterize urothelial and smooth musclecell differentiation in the retrieved bladders.Anti-Desmin antibody(monoclonal NCL-DES-DERII, clone DE-R-11, Novocastra®, Newcastle UK),which reacts with parts of the intermediate filament muscle cell proteindesmin, and Anti-Alpha Smooth Muscle Actin antibody (monoclonal NCL-SMA,clone asm-1, Novocastra®, Newcastle UK), which labels bladder smoothmuscle actin, were used as general markers for smooth muscledifferentiation. Anti-Pancytokeratins AE1/AE3 antibody (monoclonal, Cat.No.1124 161, Boehringer Mannheim®) and AntiCytokeratin 7 antibody(NCL-CK7, Clone LP5K, IgG2 b, Novocastra®, New Castle, UK) which reactagainst intermediate filaments that form part of the cytoskeletalcomplex in epithelial tissues, were used to identify urothelium.Anti-Asymmetric Unit Membrane (AUM) staining, using polyclonalantibodies, was used to investigate the presence of mammalianuroplakins, which form the apical plaques in mammalian urothelium andplay an important functional role during advanced stages of urothelialdifferentiation. Anti S-100 antibody (Sigma®; St. Louis Mo., No.IMMH-9), reacting with the acidic calcium-binding protein S-100, mainlypresent in Schwann cells and glial elements in the nervous system, wasused to identify neural tissues.

Specimens were fixed in Carnoy's solution and routinely processed forimmunostaining. High temperature antigen unmasking pretreatment withabout 0.1% trypsin was performed using a commercially available kitaccording to the manufacturer's recommendations (Sigma®, St. Louis Mo.,T-8 128). Antigen-specific primary antibodies were applied to thedeparaffinized and hydrated tissue sections. Negative controls weretreated with plain serum instead of the primary antibody. Positivecontrols consisted of normal bladder tissue. After washing withphosphate buffered saline, the tissue sections were incubated with abiotinylated secondary antibody and washed again. A peroxidase reagentwas added and upon substrate addition, the sites of antibody depositionwere visualized by a brown precipitate. Counterstaining was performedwith Gill's hematoxylin.

Trigone-Sparing Cystectomy only (Group A): The trigone-sparingcystectomy organs showed a normal histological architecture which wasconfirmed by immunocytochemical staining.

Polymer only Bladders (Group B): The polymers implanted without cellswere found to undergo a fibrovascular reaction consisting of fibroblastdeposition and extensive recruitment of inflammatory cells, includingmacrophages, and ubiquitous signs of angiogenesis at one month.Epithelial coverage was evident throughout the entire polymer. Theepithelium stained positive for the broadly reactinganti-pancytokeratins AE1/AE3, anti-cytokeratin 7, and the urotheliumspecific antiAUM. Fibrotic tissue deposition was noted at the sites ofpolymer degradation. The 2, 3 and 4 month time points showed extensionof the native submucosal and muscular layer of the trigone onto thefibrotic polymer region at the transition zone. In the 6 and 11 monthspecimens abundant connective tissue formation had replaced the fullydegraded polymer fibers of the proximally located neo-bladder region.Smooth muscle alpha actin positive cells were only scarcely evident inthis region.

Tissue Engineered Neo-Organs (Group C): The tissue engineered neoorganretrieved at one month showed complete luminal coverage with urothelium.The epithelium stained positive for the broadly reactinganti-pancytokeratins AE1/AE3, anti-cytokeratin 7, and the urotheliumspecific anti-AUM. The polymer fibers carried cell formations stainingpositive for a smooth muscle actin. An adequate angiogenic response wasevident. At two months, before the polymers underwent completebiodegradation, the muscle fibers had a spatial alignment, formingvariably sized bundles. By three months, there was complete polymerdegradation and a tri-layered structure was evident in the proximallylocated neo-bladder region, consisting of a morphologically normaluroepithelial lining over a sheath of submucosa, followed by a layercontaining multiform smooth muscle bundles. Six months postoperatively,an ingrowth of neural tissue was present for the first time as evidencedby S-100 staining. Bladders were found to have matured towards a normalhistological and phenotypic structure as evidenced by its staining withhematoxylin and eosin, trichrome, alpha smooth muscle actin, desmin,pancytokeratins AE1/AE3, cytokeratin 7 and AUM antibodies (FIGS. 5 and6). Histologically and immunocytochemically, there were no markeddifferences present between the 6 month and 11 month time pointbladders.

Example 8

Statistical Findings

Statistical evaluations were performed on the measurements using atwo-tailed Student's t-test with p-values of less than or equal to 0.05considered significant. The cystectomy only controls and polymer onlygrafts maintained average capacities of 22% and 46% of preoperativevalues, respectively. An average bladder capacity of 95% of the originalpre-cystectomy volume was achieved in the tissue-engineered bladderreplacements. The subtotal cystectomy reservoirs which were notreconstructed and polymer only reconstructed bladders showed a markeddecrease in bladder compliance (10% and 42%). The compliance of thetissue engineered bladders showed almost no difference from preoperativevalues that were measured when the native bladder was present (106%).Histologically, the polymer only bladders presented a pattern of normalurothelial cells with a thickened fibrotic submucosa and a thin layer ofmuscle fibers. The retrieved tissue engineered bladders showed a normalcellular organization, consisting of a tri-layer of urothelium,submucosa and muscle. Immunocytochemical analyses for desmin, α-actin,cytokeratin 7, pancytokeratins AE1/AE3 and uroplakin III confirmed themuscle and urothelial phenotype. S-100 staining indicated the presenceof neural structures.

The animals which had undergone the trigone-sparing cystectomy and wereclosed primarily gained a minimal amount of reservoir volume over timebut did not approach the pre-cystectomy values. The free graft polymeronly bladders had a slight increase in volume and developed fibroticneo-bladders, which had a well developed urothelial layer, but amarkedly deficient muscular architecture, and were associated with areduced compliance curve. The tissue aneroid neo-bladders were able toapproach and surpass the pre-cystectomy bladder capacities. Thecompliance of these bladders approached the pre-cystectomy values ateach time point, including the four week postoperative examination. Theretrieved tissue engineered bladders showed a normal cellularorganization, consisting of a tri-layer of urothelium, submucosa andmuscle. Immunocytochemical analysis with desmin and smooth muscle alphaactin confirmed the muscle phenotype. Pancytokeratins AE1/AE3,cytokeratin 7, and uroplakin III could be demonstrated byimmunohistochemistry, confirming the urothelial phenotype. PositiveS-100 staining suggested, that an ingrowth of neural structures into thetissue engineered bladders is possible. The tissue engineeredneobladders were able to function normally soon after implantation.Structurally and functionally, they were indistinguishable from nativebladders. Our results show, for the first time, that creation of atri-layered structure, composed of bladder muscle and urothelium invitro, is beneficial for the ultimate functional results of bladdertissue created de-novo. Our results of bladder replacement with thecell-free polymer graft are consistent with prior reports in theliterature over the last several decades regarding free grafts. Whenother materials are used as free grafts without cells, the differenthistological components may be present, but are not necessarily fullydeveloped or functional. Furthermore, the results of the cell-freepolymer bladder control group are consistent with the literature interms of graft contracture and shrinkage over time. The second controlgroup, which underwent primary closure after cystectomy, clearlyindicated that the increase in capacity in the tissue engineeredneo-bladders was due mostly to the implant and not to the naturalregenerating and elastic features of the native canine bladders. Theresults show that bladder submucosa seeded with urothelial and musclecells can form new bladder tissue which is histologically andfunctionally indistinguishable from the native bladder. This resultmaybe due to a possible maintenance of the architectural form of thebladder by the extracellular matrix regenerated by the seeded cells. Theurothelial and muscle cells seeded on the polymeric matrix appear toprevent the resorption of the graft. This technology is able to form newbladder tissue which is anatomically and functionally similar to that ofnormal bladders.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S. Patents and other referencesnoted herein for whatever reason are specifically incorporated byreference. The specification and examples should be considered exemplaryonly with the true scope and spirit of the invention indicated by thefollowing claims.

1. A method for the replacement or repair of a bladder, or portion of abladder, in a human patient in need of such treatment comprising thesteps of: a. providing a biocompatible synthetic, or natural, polymericmatrix structure in the shape of a bladder, or portion of a bladder,wherein said polymeric matrix is coated with a biocompatible andbiodegradable shape-setting material comprising a liquefiedpoly-lactide-co-glycolide copolymer; b. depositing at least one firstcell population on or in said polymeric matrix; and c. implanting theshaped polymeric matrix of step (b) into the human patient at a site ofa bladder for the formation of a laminarily organized functional bladderstructure.
 2. The method of claim 1, wherein the first cell populationis substantially a urothelial cell population.
 3. The method of claim 1,wherein the first cell population is substantially a smooth muscle cellpopulation.
 4. The method of claim 1, wherein said matrix structure inthe shape of a bladder or portion of a bladder comprises urothelialcells deposited on an inner surface of said matrix and smooth musclecells deposited on an outer surface of said matrix.
 5. The method ofclaim 1, wherein the bladder structure formed in vivo exhibits thecompliance of natural bladder tissue.
 6. The method of claim 1, whereinsaid polymeric matrix structure comprises multiple matrix layers andwherein said cell population is deposited on separate matrix layers andsaid matrix layers are combined after the deposition steps.
 7. Themethod of claim 1, further comprising depositing a second cellpopulation on or in said polymeric matrix, wherein the second cellpopulation is of a different cell type than the first cell population.8. The method of claim 7, wherein the first cell population issubstantially smooth muscle cells.
 9. The method of claim 7, wherein thesecond cell population is substantially urothelial cells.
 10. The methodof claim 7, wherein said first and second cell populations are depositedsequentially.
 11. The method of claim 1 wherein the first cellpopulation is a population of autologous cells.
 12. The method of claim1, further comprising step (d) wherein the implanted shaped polymericmatrix of step (c) is wrapped with omentum.
 13. The method of claim 1,wherein the biocompatible polymeric matrix comprises a material selectedfrom the group of materials consisting of cellulose ether, cellulose,cellulosic ester, fluorinated polyethylene, poly-4-methylpentene,polyacrylonitrile, polyamide, polyamideimide, polyacrylate,polybenzoxazole, polycarbonate, polycyanoarylether, polyester,polyestercarbonate, polyether, polyetheretherketone, polyetherimide,polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin,polyimide, polyolefin, polyoxadiazole, polyphenylene oxide,polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, copolymers thereof, and physical blendsthereof.
 14. The method of claim 1, wherein the polymeric matrixcomprises polyglycolic acid.
 15. The method of claim 1, wherein thepolymeric matrix is comprised of fibers with an interfiber distancebetween about 0 to 1000 μm.
 16. The method of claim 1, wherein thepolymeric matrix is comprised of fibers with an interfiber distancebetween about 0 to 500 μm.
 17. The method of claim 1, wherein thepolymeric matrix is comprised of fibers with an interfiber distancebetween about 0 to 200 μm.
 18. The method of claim 1, wherein the firstcell population is a population of allogenic cells.
 19. The method ofclaim 1, wherein the polymeric matrix is biodegradable.
 20. A device forthe replacement or repair of a bladder, or portion of a bladder, in ahuman comprising: an implantable, biocompatible, synthetic or natural,polymeric matrix structure in the shape of a bladder or portion of abladder having at least two separate surfaces, wherein the polymericmatrix is coated with a biodegradable shape-setting material comprisinga liquefied poly-lactide-co-glycolide copolymer, and at least one firstcell population deposited on or in said polymeric matrix to form anorganized matrix/cell construct, wherein upon implantation of saidorganized matrix/cell construct into a human, said device forms alaminarily organized functional bladder structure.
 21. The device ofclaim 20, wherein the first cell population is substantially aurothelial cell population.
 22. The device of claim 20, wherein thefirst cell population is substantially a smooth muscle cell population.23. The device of claim 20, wherein the said matrix structure in theshape of a bladder or portion of a bladder comprises urothelial cellsdeposited on an inner surface of said matrix and smooth muscle cellsdeposited on an outer surface of said matrix.
 24. The device of claim20, wherein the bladder structure formed in vivo exhibits the complianceof natural bladder tissue.
 25. The device of claim 20, furthercomprising a second cell population deposited on or in said polymericmatrix, wherein the second cell population is of a different cell typethan the first cell population.
 26. The device of claim 25, wherein thefirst cell population is substantially smooth muscle cells.
 27. Thedevice of claim 25, wherein the second cell population is substantiallyurothelial cells.
 28. The device of claim 25, wherein the first and thesecond cell populations are deposited sequentially.
 29. The device ofclaim 25, wherein said polymeric matrix structure comprises multiplematrix layers and wherein the first cell population and the second cellpopulation are deposited on separate matrix layers and said matrixlayers are combined.
 30. The device of claim 20 wherein the first cellpopulation is a population of autologous cells.
 31. The device of claim20, wherein the biocompatible polymeric matrix is formed from a materialselected from the group of materials consisting of cellulose ether,cellulose, cellulosic ester, fluorinated polyethylene,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, copolymers thereof, and physical blendsthereof.
 32. The device of claim 20, wherein the polymeric matrixcomprises polyglycolic acid.
 33. The device of claim 20, wherein thepolymeric matrix is comprised of fibers with an interfiber distancebetween about 0 to 1000 μm.
 34. The device of claim 20, wherein thepolymeric matrix is comprised of fibers with an interfiber distancebetween about 0 to 500 μm.
 35. The device of claim 20, wherein thepolymeric matrix is comprised of fibers with an interfiber distancebetween about 0 to 200 μm.
 36. The device of claim 20, wherein the firstcell population is a population of allogenic cells.
 37. The device ofclaim 20, wherein the polymeric matrix is biodegradable.